The gamma polypeptide is a small membrane protein that associates with the Na, K-ATPase. A mutation in the gene coding the subunit results in renal hypomagnesaemia associated with hypocalciuria. The conversion of a conserved glycine within the transmembrane domain to arginine (G41R) leads to misrouting of the gamma polypeptide from the plasma membrane to intracellular compartments. In the kidney, the misrouting of gamma may result in the diminution of Na,K-ATPase activity at the plasma membrane, resulting in hypomagnesaemia. However, recent results have demonstrated that misrouting of gamma does not influence the trafficking of the Na,K-ATPase. Therefore, the mechanism by which the gammaG41R mutation leads to hypomagnesaemia remains unknown. The overall goal of this proposal is to elucidate the mechanism by which a familial mutation in the gamma polypeptide results in the observed phenotype of hypomagnesaemia. Specifically the aims are: 1) To determine the mechanism by which a mutation in gamma results in renal hypomagnesaemia and hypocalciuria. This will involve the characterization of channel and ion transport properties of cultured renal epithelial cells expressing the wild type and mutant gamma subunits. 2). To determine, using gene chip technology, if expression of the T subunit influences the expression of other genes. We will characterize the gene expression pattern of cultured renal epithelial cells expressing the wild type and gammaG41R subunits. It is possible that the observed phenotype of the gammaG41R mutation is a result of the induction of genes not directly related to ion transport. For example, the accumulation of unfolded proteins in the ER may activate pathways to induce programmed cell death. Thus, the observed phenotype may be a result of the apoptotic death of specific tubule cells resulting in the disruption of the transport properties of the nephron. These studies will elucidate the physiological role of the gamma subunit in renal function, and provide important information concerning the etiology of the hypomagnesaemia associated with the gamma G41R mutation. In addition, these results may identify essential renal pathways responsible for degrading or refolding non-native proteins or pathways that influence stress induced apoptosis.